Histo-blood group Lewis genotyping from human hairs and blood

The expression of histo-blood group Lewis antigens is determined by the Lewis-type alpha 1-->3/4fucosyltransferase (Le enzyme) encoded by Fuc-TIII gene (Le gene). The genotyping of Le genes by the PCR-RFLP methods established recently and partly modified in this study was found to be useful not only for determining the genuine Lewis blood types of samples such as human hairs and blood stains but also for distinguishing non-genuine Lewis-negative phenotypes frequently observed in pregnant women from genuine ones. The availability of the present PCR-RFLP methods for the paternity tests was also discussed.     

A novel mutation in the coding sequence of the FY*B allele of the Duffy chemokine receptor gene is associated with an altered erythrocyte phenotype

The Duffy blood group system is of clinical and biological significance. Antibodies to Duffy antigens are responsible for some cases of transfusion incompatibility and newborn hemolytic disease. The Duffy protein is a receptor for the Plasmodium vivax erythrocyte-binding protein and is also a receptor for various chemokines (thus renamed Duffy Antigen Receptor for Chemokines [DARC]). The two Duffy polymorphic antigens, Fya and Fyb (coded by the FY*A and FY*B alleles), are present on erythrocyte membranes. The Fy(a-b-) phenotype is the predominant one in populations of black people and also occurs in other populations, including some non-Ashkenazi Jewish groups. The Fy(a-b-) phenotype has been associated with a mutation in the FY*B promoter at the GATA box that abolishes the expression of erythrocyte Duffy protein. We describe here a novel mutation, present in the FY*B coding sequence (271C --> T), that is associated with some Fy(b-) phenotypes among non-Ashkenazi Jews and among Brazilian blacks. The mutation is present in Fy(b-) individuals, who have wild-type FY*B GATA and carry the previously described 304G --> A substitution. The 271C --> T and 304G --> A can be identified by restriction enzyme-generated restriction fragment length polymorphisms. The 271C --> T substitution represents a considerable change in chemical nature (Arg91 --> Cys), one which may affect the antigenic determinants of DARC, and thus be of clinical significance. The mutation may have implications for some physiological roles of DARC and be of interest in malaria research and in studies of population genetics.     

Molecular basis for the high-incidence antigens of the Kell blood group system

BACKGROUND: The Kell blood group system is complex, containing at least 21 antigens. Some antigens are organized in five allelic sets; other, mostly high-incidence antigens, may be independently expressed. In this study, the molecular basis of five high-incidence antigens in the Kell system are described. STUDY DESIGN AND METHODS: Genomic DNA sequences from K:-12 (KEL:-12), K:-18 (KEL:-18), K:-19 (KEL:-19), K:-22 (KEL:-22), and TOU-(KEL:-26) persons were sequenced and compared to those from persons with a common Kell phenotype. RESULTS: The various Kell phenotypes are due to point mutations that encode amino acid substitutions. In KEL:-18, two mutations in the same codon were noted. In the various phenotypes, the following KEL mutations were noted: in KEL:-12: A1763G, His548Arg; in KEL:-18: C508T and G509A, Arg130Trp and Arg130Gln; in KEL:-19: G1595A, Arg492Gln; in KEL:-22: C1085T, Ala322Val; and in TOU-:G1337A, Arg406Gln. A son of one of the two people with the TOU-phenotype was heterozygous, and he also had the G1337A mutation. CONCLUSION: The high-incidence antigens of the Kell blood group system are characterized by point mutations leading to amino acid substitutions. The KEL:-18 phenotype could be due to either of two point mutations in the same codon replacing arginine with tryptophan or glutamine. TOU was confirmed as a Kell system antigen, and the inheritance of the mutation was demonstrated.     

Alcohol intake, Lewis phenotypes and risk of ischemic heart disease. The Copenhagen Male Study

In the Copenhagen Male Study we found an increased risk of ischaemic heart disease (IHD) in men with the Lewis phenotype Le(a-b-). This study investigated whether, within the group of Le(a-b-) men, any conventional risk factors modified their increased risk. Three thousand, three hundred and eighty-three men aged 53 to 75 years were examined in 1985/86 and their morbidity and mortality over the next four years recorded. Three hundred and forty-three men with cardiovascular diseases were excluded at baseline. Potential risk factors examined were: alcohol consumption, physical activity, tobacco smoking, serum cotinine, serum lipids, body mass index, blood pressure, hypertension, non-insulin dependent diabetes mellitus and social class. In eligible men with Le(a-b-), N = 280 (9.6%), alcohol was the only risk factor associated with risk of IHD. There was a significant inverse dose-effect relationship between alcohol consumption and risk. The age-adjusted p-values of trend tests were for risk of non-fatal + fatal IHD: p = 0.03; for risk of fatal IHD: p = 0.02. In eligible men with other phenotypes, N = 2,649 (90.4%) only a limited and non-significant negative association with alcohol. In Le(a-b-) men, a group genetically at increased risk of IHD, the risk was strongly and significantly negatively correlated with alcohol consumption.     

Evolution of precipitates in lead-implanted aluminum: A backscattering and channeling study

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.     

Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene

All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins.     

Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): possible involvement of the peroxisome proliferator-activated receptor alpha (PPAR alpha)

Although several variant alleles at the human NAT1 gene locus have been reported, their relationship to phenotypic variations in NAT1 function remains unclear. We have used in-vivo and invitro phenotyping tests, along with PCR-based cloning and heterologous expression, to investigate the extent of variation in NAT1 function and to characterize novel allelic variants at the NAT1 gene locus. The NAT1-selective substrate p-aminosalicylic acid (PAS) was used as a probe for NAT1 function. In-vivo PAS acetylation rates were estimated by determining the ratio of PAS to N-acetylated PAS (AcPAS) in urine and plasma following the oral ingestion of Nemasol Sodium. Excluding outliers, a 65-fold variation in the urinary AcPAS:PAS ratio was observed (n = 144), while a 5.6-fold variation in the plasma AcPAS:PAS ratio was seen in a subset (n = 19) of this sample. Urinary and plasma ratios correlated moderately (r = 0.74, p < 0.0005). One individual (case 244) had a marked impairment of PAS N-acetylation, with 10-fold lower urinary and plasma AcPAS:PAS ratios compared with other subjects. Biochemical investigations in whole blood lysates from case 244 suggested a NAT1 kinetic defect, with a 20-fold increased apparent K(m) for PAS and a 90-fold decreased Vmax for AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from seven other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT1 x 14 and NAT1 x 15, in case 244, as well as one variant, NAT1 x 11, which has been observed in previous investigations. NAT1 x 14 contained a missense mutation (G560-->A) that is predicted to change a single amino acid (Arg187-->Gln), as well as two 3' non-coding region mutations (T1088-->A and C1095-->A) that have previously been observed in the NAT1 x 10 allelic variant. NAT1 x 15 had a single nonsense mutation (C559-->T; Arg187-->stop) and, thus, encodes a truncated protein. The activity of recombinant NAT1 14 mirrored the defective enzyme function in whole blood lysates from case 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on the other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functionally silent. The frequencies of NAT1 x 11, NAT1 x 14 and NAT1 x 15 were 0.021, 0.028 and 0.014 (n = 288 alleles), respectively, suggesting that they are relatively rare in our predominantly Caucasian sample.     

Expression, purification and kinetic properties of human recombinant phospholipase C delta 3

Fucosylated histo-blood group antigens such as Lewis b, Lewis Y, and H accumulate in colon carcinoma and this is accompanied by a clear increase in alpha(1-2)fucosyltransferase activity, a key enzyme for the biosynthesis of these antigens. Yet the biological significance of alpha(1-2) fucosylated structures is not well defined. We have transfected a poorly tumorigenic rat colon carcinoma cell line with the human H blood group alpha(1-2)fucosyltransferase cDNA. This resulted in cell surface expression of H antigens with a concomitant decrease of sialic acid substituted and free beta-galactosides. Immunoprecipitation experiments showed that H antigens were essentially borne by variants of CD44 carrying amino acid sequences encoded by exon v6. The transfected cells showed increased motility in a wound healing assay, without changing their proliferation rates. Parental and control cells transfected with an empty vector formed small tumors that always regressed after 30 days when injected subcutaneously to syngeneic rats. In contrast, alpha(1-2)fucosyltransferase transfectants were able to form progressive tumors. Increased tumorigenicity was also visible in nude mice. These results demonstrate that alpha(1-2)fucosylated antigens contribute directly to aggressiveness of colon carcinoma cells. This could occur by altering a function of CD44 variants.     

Lutheran antigens, CD44-related antigens, and Lutheran regulatory genes

The Lutheran (Lu) blood group antigens are a family of human erythrocyte antigens which reside on two closely-related erythrocyte integral membrane proteins. Sixteen Lutheran or so-called para-Lutheran antigens have thus far been described, and human antisera to many of them have been shown to immunoblot two proteins, of 78 and 85 kDa. Lu cDNA encodes an integral membrane protein of 597 amino acids that is a member of the Ig superfamily. Lu proteins comprise five Ig superfamily domains, along with a single transmembrane domain and a cytoplasmic domain of about 60 amino acids. The two proteins seen in biochemical studies of red cell membranes appear to be derived from 2 mRNA species that differ only in their 3' ends, suggesting that they arise from alternate splicing of a single preRNA. Three genetic backgrounds for the Lu(a-b-) [Lu null] phenotype have been described. A recessive Lu null phenotype is rarely observed as a result of homozygosity for two amorphic LU alleles. However, the most common Lu(a-b-) phenotype appears to be caused by an independently segregating, dominant gene, designated In (Lu), which inhibits expression of all Lutheran antigens to nearly undetectable levels. This gene also affects the expression of other cell surface proteins and blood group antigens that are genetically unlinked to the Lutheran locus, including CD44 and MER2. CD44, a member of the cartilage link family of proteins, bears the In and AnWj blood group antigens. A widely distributed protein CD44 is expressed at normal levels on all tissues except erythrocytes in the presence of the In (Lu) gene. A second Lutheran regulatory gene, XS2, is responsible for the third Lu(a-b-) phenotype, which exhibits an X-linked inheritance pattern. The XS2 gene down-regulates but does not abolish expression of LU genes and does not affect expression of CD44.     

Expression of the beta 3-adrenoceptor gene polymorphism (Trp64Arg) in obese diabetic and non-diabetic subjects

1. A missense mutation (Trp64Arg) in the beta 3-adrenoceptor (beta 3-AR) gene has been associated with weight gain, insulin resistance and earlier-onset non-insulin-dependent diabetes mellitus (NIDDM), but the strength of these associations varies considerably between populations and the functional significance of Trp64Arg remains unclear. 2. The Trp64Arg mutation was investigated in obese NIDDM (n = 50) and obese non-diabetic (n = 53) subjects by polymerase chain reaction (PCR) amplification of genomic DNA and digestion of the 210 bp product by BstOI. The Arg allele was found in 22.3% of all subjects, but there were no homozygotes for the mutation. Non-diabetic subjects heterozygous for the mutation were more obese and Trp/Arg diabetics had a slightly younger age of onset of NIDDM (47 vs 51 years, respectively), but there were no significant differences in mutation frequency between the two groups. Metabolic parameters (e.g. fasting lipids and glycaemic control) were similar among diabetic subjects with and without the Trp64Arg mutation. 3. In conclusion, the frequency of the Trp64Arg mutation of the beta 3-AR was higher in this obese population compared with some previous studies, but there was no evidence that Trp64Arg confers an increased susceptibility to NIDDM among obese insulin-resistant subjects or that diabetics with the mutation fare worse in terms of lipid or glucose metabolism.     

A common mutation in the methylenetetrahydrofolate reductase gene (C677T) increases the risk for deep-vein thrombosis in patients with mutant factor V (factor V:Q506)

Hyperhomocysteinemia is a frequent risk factor for deep-vein thrombosis. A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) is responsible, in the homozygous state, for decreased enzyme activity and mild hyperhomocysteinemia and is associated with increased risk for cardiovascular disease. We studied the prevalence of C677T MTHFR in 77 patients with deep-vein thrombosis and in 154 age- and sex-matched healthy control subjects. In the same individuals, we also evaluated the frequency of the coexistence of C677T MTHFR with mutant factor V:Q506, a common risk factor for deep-vein thrombosis. Sixteen patients (20.8%) and 35 control subjects (22.7%) were homozygous for the C677T MTHFR mutation (odds ratio [OR] = 0.8, 95% confidence interval [CI] = 0.4-2.0). Sixteen patients (20.8%) and 4 control subjects (2.6%) had factor V:Q506; of them, 10 patients and 3 control subjects had isolated factor V:Q506 (adjusted OR = 6.3, 95% CI = 1.6-25.3) and 6 patients and 1 control subject also had C677T MTHFR (adjusted OR = 17.3, 95% CI = 2.0-152.9). The OR for the coexistence of the two mutations was 65% to 75% higher than the expected joint effect calculated by either an additive (OR = 6.0) or multiplicative (OR = 4.4) model. The homozygous C677T mutation of MTHFR per se is not a risk factor for deep-vein thrombosis but increases the risk associated with factor V:Q506. Due to the high prevalence of C677T MTHFR, it is likely that previous studies, which did not look for this mutation, overestimated the relative risk of thrombosis associated with factor V:Q506 alone.     

Two different allelic mutations in a Finnish family with lecithin:cholesterol acyltransferase deficiency

Lecithin:cholesterol acyltransferase (LCAT) deficiency is a genetic disorder associated with low levels of serum HDL cholesterol. The proband of the Finnish LCAT-deficient family had corneal opacities, proteinuria, anemia with stomatocytosis, low serum HDL cholesterol (0.27 mmol/L), and low LCAT activity. Sequence analysis of his LCAT gene revealed compound heterozygosity for two different mutations: a C insertion in exon 1 between nucleotides 932 and 937 and a C-to-T point mutation in exon 6 at position 4976. The C insertion in exon 1 is predicted to result in premature termination and a truncated polypeptide containing only 16 amino acids. The C-to-T point mutation in exon 6 substitutes cysteine for arginine at residue 399. The functional significance of the Arg399-->Cys mutation was examined by expressing the mutated and wild-type LCAT cDNAs in COS cells. COS cells transfected with mutated and wild-type cDNAs showed comparable levels of mature LCAT mRNA. However, LCAT activity in the cell media of COS cells transfected with the mutant LCAT cDNA was significantly lower than that of COS cells transfected with the wild-type cDNA (1.4% versus 12.0% cholesterol esterified, respectively). A polymerase chain reaction-based duplex assay, in which both mutations can be detected simultaneously, was used for preliminary screening of Finnish subjects with serum HDL levels below 0.9 mmol/L; two additional individuals heterozygous for the Arg399-->Cys mutation were identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a non-classical mutant of the DNA recognition subunit of the type I restriction endonuclease R.EcoR124I

We have used deletion mutagenesis and PCR-based misincorporation mutagenesis to produce a collection of mutations in the central conserved region of the DNA binding subunit of the type IC restriction endonuclease EcoR124I. It has been proposed that this domain is involved in protein-protein interactions during the assembly of the endonuclease. While a large percentage of these mutations gave a classical Res- Mod- phenotype, one mutant was isolated with a nonclassical Res- Mod+ phenotype. The loss of restriction activity, but retention of the ability to modify indicates that this mutation cannot affect DNA binding and must alter the assembly of the endonuclease in such a way as to prevent DNA cleavage but allow methylation. This mutant resulted from a single amino acid change Trp212-->Arg. The location of the single amino acid change is at the border of the central conserved region and the second target recognition domain (TRD2) and suggests that this region is extremely important for the assembly of the methylase with the HsdR subunit into a functional restriction endonuclease.     

Loss of activity of plasma platelet-activating factor acetylhydrolase due to a novel Gln281-->Arg mutation

The prevalence of plasma platelet-activating factor (PAF) acetylhydrolase deficiency was investigated in 477 healthy Japanese individuals and 985 patients with various cardiovascular diseases. The genotype for this enzyme with regard to a G994-->T mutation (MM, normal; Mm, heterozygote; mm, mutant homozygote) was determined by an allele-specific polymerase chain reaction in 80 subjects shown to have no or low plasma activity (<10 nmol/min/ml). In 72 subjects, the genotype was consistent with plasma enzyme activity; 44 individuals with no activity were mm, and 28 with low activity were Mm. However, eight subjects with the MM genotype showed plasma enzyme activities of <10 nmol/min/ml. Determination of the DNA sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects revealed a previously unidentified A1001-->G missense mutation, resulting in a Gln281-->Arg substitution, in a 72-year-old woman with coronary artery disease, essential hypertension, and no plasma enzyme activity. Site-directed mutagenesis in vitro showed that the corresponding recombinant mutant protein lacked PAF acetylhydrolase activity. Thus, the Gln281-->Arg substitution appears responsible for the loss of plasma PAF acetylhydrolase activity.     

Five novel mutations of the protein S active gene (PROS 1) in 8 Norman families

To further elucidate the molecular basis for hereditary thrombophilia, we screened the protein S active gene in 11 families with type I deficiency, using a strategy based on denaturing gradient gel electrophoresis (DGGE) of all the coding sequences. Fragments with an abnormal DGGE pattern were sequenced, and 5 novel mutations were identified in 8 families. The mutations were a 7-nucleotide deletion in exon II, a 4-nucleotide deletion in exon III, a T insertion in exon VII, a C to T transition transforming Leu 259 into Pro and a T to C transition transforming Cys 625 into Arg in 4 families. These mutations were the only sequence variations found in the propositus' gene exons and co-segregated with the plasma phenotype. A total of 28 members of these 8 families were heterozygous for one of the 5 mutations. Twenty-four (58,5%) of the 41 deficient subjects over 18 years of age had clinical thrombophilia, whereas the 13 subjects under 18 were asymptomatic. Of the 28 subjects, 6 (21,5%) were also found to bear the factor V Arg 506 Gln mutation.     

An adenosine deaminase (ADA) allele contains two newly identified deleterious mutations (Y97C and L106V) that interact to abolish enzyme activity

Genetic deficiency of the purine salvage enzyme adenosine deaminase (ADA) results in varying degrees of immunodeficiency, ranging from neonatal onset Severe Combined Immunodeficiency (SCID) to an adult onset immunodeficiency disorder. Multiple different mutations have now been identified in these immunodeficient patients. Additional mutations, initially identified in healthy individuals, abolish ADA in erythrocytes but retain 10-80% of activity in non-erythroid cells ('partial deficiency mutations'). In general, severity of disease correlates inversely with the amount of residual ADA expressed by the mutant enzymes and directly with the accumulation of the toxic metabolites deoxyATP and deoxyadenosine. We report two newly identified mutations (Y97C and L106V), both carried on the same allele of an immunodeficient patient who was diagnosed prenatally and successfully transplanted with haploidentical bone marrow. Based on the ability of mutant cDNAs to express ADA in vitro , the L106V mutation resulted in activity similar to 'partial' mutations (30% of normal) while the Y97C mutation resulted in detectable but markedly reduced activity (1.5% of normal). However, the presence of both mutations on the same allele virtually abolished detectable enzyme activity. Analysis of the crystallographic structure of ADA to understand the marked deleterious effect of the Y97C mutation suggested a previously unappreciated role of salt bridges in the catalytic mechanism of ADA. The patient was also heteroallelic for a previously described deletion of the promoter and exon 1. Testing of additional patients in whom we had not identified a mutation on the second allele revealed presence of this deletion in three of four patients tested. This deletion is therefore relatively common, accounting for 10% of almost 100 chromosomes studied by this and other laboratories, but is easily missed by currently used methods of mutation detection. Lastly, the finding of two mutations on the same allele that interact to reduce residual enzyme function emphasizes hazards in evaluating potential genotype-phenotype correlations in individuals analyzed only for the presence of single specific mutations.